Cyclosporin binding to a protein component of the renal Na(+)-D-glucose cotransporter.
The immunosuppressive and nephrotoxic agent cyclosporin binds to a renal polypeptide with an apparent molecular weight of 75,000 which has been identified as a component of the renal Na(+)-D-glucose cotransporter (Neeb, M., Kunz, U., and Koepsell, H. (1987) J. Biol. Chem. 262, 10718-10729). The same Mr 75,000 polypeptide was covalently labeled with the D-glucose analog 10-N-(bromoacetyl)amino-1-decyl-beta-D-glucopyranoside and with the cyclosporin analog N epsilon-(diazotrifluoroethyl)benzyl-D-Lys8- cyclosporin (CSDZ). CSDZ labeling was decreased when the brush-border membrane proteins were incubated with monoclonal antibodies against the Na(+)-D-glucose cotransporter. In the presence of 145 mM Na+, CSDZ labeling was decreased by D-glucose (1 microM, 1 mM, or 100 mM) and by phlorizin (100 or 500 microM). In the absence of Na+, CSDZ labeling was distinctly increased by 50 microM phlorizin and was slightly increased by 1 mM D-glucose, whereas CSDZ labeling was decreased by 50 microM phloretin and by 500 microM phlorizin. Furthermore, Na(+)-dependent high affinity phlorizin binding to the Na(+)-D-glucose cotransporter was competitively inhibited by cyclosporin A (Ki = 0.04 microM) while Na(+)-D-glucose cotransport was not influenced. The data suggest that a part of the cyclosporin binding domain on the Na(+)-D-glucose cotransporter is identical to the phloretin binding domain of the high affinity phlorizin binding site. While phloretin or the phloretin moiety of phlorizin may directly displace cyclosporin, interaction of D-glucose or of the D-glucose moiety of phlorizin with the transporter may alter the conformation of the cyclosporin binding site and this conformational change may be modulated by Na+.
Ziegler
K
K
Institut für Pharmakologie und Toxikologie, Justus-Liebig Universität Giessen, Federal Republic of Germany.
Frimmer
M
M
Fritzsch
G
G
Koepsell
H
H
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D003524:Cyclosporins / Q000378:metabolism* / Q000494:pharmacology; D007672:Kidney Cortex / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008871:Microvilli / Q000378:metabolism*; D008961:Models, Structural; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010695:Phlorhizin / Q000378:metabolism; D011485:Protein Binding; D013552:Swine
D000345:Affinity Labels; D001386:Azides; D003524:Cyclosporins; D046911:Macromolecular Substances; D009004:Monosaccharide Transport Proteins; C063121:N(epsilon)-(diazotrifluoroethyl)benzoyl-Lys(8)-cyclosporin; D010695:Phlorhizin